Gene activity is regulated at multiple levels. Thus, tissue-specific gene regulations is controlled at a level above the gene promoter by interactions between cis-acting DNA elements (e.g., enhancers) with factors (e.g., transcription factors) that activate or repress gene expression. In contrast, what may be called domain gene regulation acts at a higher level, to control the expression of multiple contiguous genes. X-chromosome inactivation is perhaps the best known and most dramatic example of this level of regulation. The XG blood group is chosen here as a model system for the study of domain gene regulation: two tightly linked genes on the X chromosome are known to be co-regulated, XG and MIC2; both genes escape X-chromosome inactivation; they are coordinately expressed in red blood cells. The experiments proposed are designed to characterize a recently isolated gene, BM22, that we consider to be an excellent candidate for being the XG structural locus. We propose further to clone and to characterize the cis-acting regulatory element XGR that is postulated to control the coordinate expression of XG and MIC2. Our specific experimental aims are the following: In one set of experiments, we propose to analyze the RNA expression of BM22 in adult venous and umbilical cord bloods from Xg(a+) and Vg(a-) individuals employing a quantitative RT-PCR method; to raise antibodies to BM22 and determine whether they react differentially to Xg(a+) and Xg(a-) red cells; and to transfect BM22 into cultured cells and determine whether Xg antibodies react differentially to transfected untransfected cells. In the second set, we propose to localize the postulated regulatory locus XGR by analysis of DNA polymorphisms segregating in several unusual families in which recombination between XGR and MIC2 is known to have occurred; to employ S1 nuclease protection assays on RNA from transfected cells to detect differential transcriptional activation of the BM22 and MIC2 promoters in reporter gene constructs into which the XGR elements from either homozygous Xga or homozygous Xga individuals have been subcloned; and to sequence the XGR elements from homozygous Xga and homozygous Xg individuals and determine by transfection studies whether mutations that convert each element into the alternate allelic form can cause a switch in the differential transcriptional activation of the BM22 and MIC2 promoters.